Letter
pubs.acs.org/NanoLett
Ratiometric Tension Probes for Mapping Receptor Forces and
Clustering at Intermembrane Junctions
Victor Pui-Yan Ma,† Yang Liu,† Lori Blanchfield,‡ Hanquan Su,† Brian D. Evavold,‡ and Khalid Salaita*,†,§
†
Department of Chemistry, Emory University, Atlanta, Georgia 30322, United States
Department of Microbiology and Immunology, Emory University, Atlanta, Georgia 30322, United States
§
Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia
30322, United States
‡
S Supporting Information
*
ABSTRACT: Short-range communication between cells is required for the survival of multicellular organisms. One mechanism
of chemical signaling between adjacent cells employs surface displayed ligands and receptors that only bind when two cells make
physical contact. Ligand−receptor complexes that form at the cell−cell junction and physically bridge two cells likely experience
mechanical forces. A fundamental challenge in this area pertains to mapping the mechanical forces experienced by ligand−
receptor complexes within such a fluid intermembrane junction. Herein, we describe the development of ratiometric tension
probes for direct imaging of receptor tension, clustering, and lateral transport within a model cell−cell junction. These probes
employ two fluorescent reporters that quantify both the ligand density and the ligand tension and thus generate a tension signal
independent of clustering. As a proof-of-concept, we applied the ratiometric tension probes to map the forces experienced by the
T-cell receptor (TCR) during activation and showed the first direct evidence that the TCR-ligand complex experiences sustained
pN forces within a fluid membrane junction. We envision that the ratiometric tension probes will be broadly useful for
investigating mechanotransduction in juxtacrine signaling pathways.
KEYWORDS: Receptor clustering, intermembrane junction, T-cell, artificial antigen presenting cell, molecular tension probe,
immunological synapse
M
lacking. This is due to the absence of methods to measure
the mechanical forces experienced by ligand−receptor complexes during active signaling at the cell membrane.
The archetypal example highlighting the complex interplay
between ligand-induced binding, receptor clustering, and
mechanical coupling is illustrated by the T-cell receptor
(TCR).10 A polarized and crawling T-cell constantly scans
the surfaces of antigen-presenting cells (APCs) in search of
foreign peptides that are bound to the major histocompatibility
complex (pMHC).11,12 Upon TCR engagement and activation
by their cognate pMHC ligand, the receptors form signaling
microclusters that initiate T-cell activation cascades leading to
embrane receptors are ubiquitous in Nature and play a
central role in transferring chemical information across
the cell membrane.1 The first step in the majority of signal
transduction cascades, ranging from growth factor signaling2,3
to T-cell activation,4−6 is ligand-induced dimerization and
oligomerization of receptors. These higher-order clusters of
receptors often function as a signaling complex for signal
amplification, diversification, and in some cases serve to
facilitate receptor internalization and signal degradation.7
Interestingly, receptor oligomers are typically coupled to the
cytoskeleton, which offers an active scaffold for receptor
translocation and organization, thus further fine-tuning signaling circuits.8 Given the role of the cytoskeleton in force
transmission and generation,9 it seems intuitive to conclude
that ligand-induced receptor clustering is intimately linked with
mechanotransduction but evidence for this connection is
© 2016 American Chemical Society
Received: May 4, 2016
Revised: May 14, 2016
Published: May 18, 2016
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Scheme 1. Schematic Representation of Gold Nanoparticle-Based Ratiometric Tension Probes
Ca 2+ flux and cytokine production. During activation,
lymphocyte function-associated antigen 1 (LFA-1) receptors
bind intercellular adhesion molecule 1 (ICAM-1) on the APC,
thus facilitating the formation of a stable intercellular junction.8
Within this specialized junction, the T-cell cytoskeleton
associates with TCR and LFA-1 and sorts these receptors
into distinct concentric zones. TCR microclusters undergo
continuous translocation and become spatially organized in a
structure known as the central supramolecular activation cluster
(cSMAC), while LFA-1/ICAM-1 are reorganized into a ringlike
structure surrounding the cSMAC.4,13
The migratory nature of T-cells and the central role of the
cytoskeleton in TCR activation14,15 suggest a fundamental
connection between receptor oligomerization, signaling, and
mechanotransduction. Several lines of evidence support an
important role of mechanical force in T-cell activation and
signaling.16,17 First, Reinherz, Lang, and co-workers showed
that TCR is a mechanosensor that responds to externally
applied piconewton (pN) forces from an optical tweezer.18
This concept is further supported by Li and co-workers who
demonstrated micropipette-induced shear forces can activate Tcells,19 and recently by our optomechanical actuator (OMA)
nanoparticles which, upon stimulation of NIR light, are able to
mechanically activate T-cells.20 In addition, TCR undergoes
distinct conformational transitions upon experiencing pN
forces transmitted through the pMHC antigens.21 Second,
biomembrane force probe experiments by Zhu, Evavold, and
colleagues showed that TCR-pMHC bond lifetime (1/koff) is
best correlated to antigen potency when 10−15 pN forces are
applied to the TCR-pMHC complex.22 Third, T-cells apply
nanonewton (nN) traction forces to deform micron-sized
PDMS pillars within ∼5 min of activation.23 Lastly, restriction
of TCR transport with diffusion barriers upregulates TCR
signaling, possibly due the increased forces on the TCR-pMHC
complex and its altered spatial organization.24 Still, whether the
interplay of mechanics, clustering, and chemical signaling
influences T-cell function remains a longstanding question
due to the lack of tools to quantify nanoscale mechanics of Tcells.
To investigate how physical inputs regulate or couple to
signal transduction in living cells, we invented molecular
tension fluorescence-based microscopy (MTFM), which maps
pN receptor forces generated by cells using fluorescence
imaging with high spatial (∼200 nm) and temporal (∼ms)
resolutions.25,26 Typically, MTFM probes consist of an
extendable “spring” flanked by a fluorophore and quencher
and immobilized on a surface. The probe is decorated with a
ligand of interest, and is highly quenched in the resting
conformation (>90% quenching efficiency). When cell surface
receptors engage their ligand on the MTFM probe and apply
sufficient mechanical load to extend the “spring” then the dye is
dequenched, which leads to significant enhancement in
fluorescence. Second generation MTFM probes have further
improved the dynamic force range,27 sensitivity28,29 and
stability.30−33 We recently developed a gold nanoparticle
(AuNP)-based DNA tension probe to directly image the
TCR-pMHC forces on immobilized ligands and revealed that
T-cells may harness mechanics for enhanced antigen sampling
and discrimination.34
One remaining limitation for MTFM is that probes are
immobilized onto a solid substrate, which is used for several
reasons. First, it limits the lateral mobility of the ligand and thus
the probe reports the integrated forces transmitted in the lateral
and perpendicular directions through the ligand−receptor
complex. Immobilized MTFM probes provide mimics of the
cell−extracellular matrix interactions, where ligands are physically affixed onto an immobile scaffold.35 Second, by
maintaining a constant probe density, the donor fluorescence
intensity can be directly used to determine the quenching
efficiency (QE) using the following relation: QE =
1 − (IDA/ID), where IDA is the donor fluorescence under zero
force condition and ID is the donor fluorescence in the presence
of applied forces. In this way, a nonfluorescent acceptor
(quencher) can be used, which reduces bleed-through,
improves signal-to-noise ratio, and also frees up additional
fluorescence channels for live cell imaging of protein translocation and signaling activity. An alternate immobilization
strategy would be to tether ligands onto laterally fluid surfaces
mimicking the plasma membrane. In this scenario, it remains
unknown whether MTFM probes on these surfaces would
experience sufficient pN tension to generate detectable signal.
In order to study mechanical forces at cell−cell junctions where
ligands and receptors are allowed to diffuse laterally, it is
necessary to introduce new methods that effectively integrate
MTFM probes with fluid membranes.
One approach to mimic the cell−cell junction is the hybrid
cell−supported lipid bilayer platform. The supported lipid
bilayer (SLB) is comprised of phospholipids that self-assemble
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Figure 1. (A) Illustration of the contact zone between a biotinylated microparticle and a DNA tension probe surface anchored to a supported lipid
membrane. (B) Representative brightfield, RICM, Cy3B, A488, and tension density images showing the contact zone of a microparticle (diameter =
5 μm) that binds to SLB tension probes (F1/2 = 4.7 pN). Scale bar = 5 μm. (C) Plot displaying line scan of Cy3B and A488 channels for the
microparticle shown in panel B. The intensity (I) is normalized to the background regions lacking the microparticle (I0). (D) Plot overlaying the line
scan profile of fluorescence and RICM channels and demonstrating their spatial colocalization. (E) Bar graph showing the tension density of
microparticles engaged to tension probe SLBs (F1/2 = 4.7 pN) and control SLB surfaces decorated using DNA duplexes (n = 20 for each sample,
error bar represents SD of the data) within the microparticle-SLB contact zone. (F) Representative FRAP images showing recovery after 90 s. Scale
bars = 10 μm. (G) Representative FRAP recovery plots for Cy3B and A488 channels. Solid lines represent the fit made using the following equation
I(t) = A(1 − e‑tτ). The lateral diffusion coefficient (D) is calculated by D = w2/4t1/2,54 where w is the radius of the Gaussian bleaching area; t1/2 is the
time for 50% recovery obtained from the fit. The values used for the calculation were w = 10.4 μm (for both channels); t1/2 = 26.5 s (Cy3B) and 27.9
s (A488).
onto a solid substrate. These lipids form a fluid bilayer that can
be chemically decorated with a variety of biomolecules, and
thus the SLB displays chemical and physical properties that
resemble those of the cellular plasma membrane.36,37
Importantly, when a living cell encounters an SLB displaying
appropriate ligands, the cell surface receptors bind, oligomerize,
and laterally translocate across the cell−SLB junction. Therefore, this platform provides a powerful tool to reconstitute
juxtacrine signaling pathways such as immunological synapse
formation during T-cell signaling,38−42 receptor clustering in Ecadherin,43 EphA2−EphrinA1,44,45 Notch−Delta,46 and integrin−RGD47−49 systems. Taking advantages of the cell−SLB
platform, we aimed to tether MTFM tension probes onto an
SLB to enable the study of receptor mechanics during active
signaling at model cell−cell junctions.
To generate SLB-tethered tension probes, we first designed a
DNA hairpin structure that places a fluorophore-quencher pair
(Cy3B-BHQ2) in close proximity. DNA hairpins are
immobilized onto a AuNP that further quenches the Cy3B
fluorescence. The AuNP-tension probe is in turn immobilized
onto a SLB using biotin−streptavidin and is further functionalized with a ligand. DNA hairpins unfold in response to
mechanical strain, which separates the fluorophore from the
molecular quencher and the AuNP surface. The magnitude of
force needed to unfold the hairpin can be estimated from the
F1/2, which is the force that leads to 50% probability of
unfolding at equilibrium.50 Note that the loading rate and the
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Figure 2. (A) Representative time-lapse images (RICM, Cy3B, A488, and tension density) showing the first 15 min of CD4+ T-cell engagement
with the CD3-tension probes anchored onto an SLB. (B) The kymographs display tension density and the A488 intensity as a function of time
within the three regions of interest (lines in the tension density channel from (A). Scale bar = 5 μm.
duration of forces are unknown and thus the F1/2 serves as a
lower bound estimate of the applied force. Because of the dual
quenching of the dye by a molecular quencher (FRET)51 and
the AuNP via Nanometal Surface Energy Transfer (NSET),52
this construct provides a 10-fold improvement in sensitivity
over the existing MTFM probes.27,28 To distinguish fluorescence enhancement due to clustering from that of tension,
we introduced a second fluorophore (Alexa Fluor 488,
abbreviated A488 thereafter) to report on relative probe
density (Scheme 1). The size of AuNP-tension probes is
estimated to be 47.4 ± 1.8 nm from dynamic light scattering
(DLS) (Figure S1). Each particle is labeled with 38 ± 1
hairpins as determined by fluorescence calibration (Figure S2).
To demonstrate that the ratiometric tension probe can
reliably report on clustering and mechanics, we employed a
model system to mimic the cell−SLB junction. In this system,
tension probes (F1/2 = 4.7 pN, see Table S1 for DNA
sequences) were immobilized onto a SLB and decorated with
streptavidin that engaged biotinylated microparticles (diameter
= 5 μm) (Figure 1A). It has been shown that the junction
between a spherical particle and planar SLB generates
interfacial tension across the ligand−receptor complexes
(biotin−streptavidin) bridging the particle to the SLB.53 This
is due to the geometric mismatch between the particle and
planar surface. We anticipated accumulation of streptavidin as
well as the extension of a subset of DNA hairpins bridging the
particle and the surface within the contact zone. Incubation of
biotinylated microparticles onto the tension probe-SLB surface
generated a strong interference pattern in reflection interference contrast microscopy (RICM), indicating close nanoscale
contact between the particle and surface (Figure 1B). This
interaction led to a drastic increase in fluorescence in both the
Cy3B and the A488 signals (Figure 1B). Line scan analysis
across the microparticles showed a maximum of ∼14-fold
increase in Cy3B fluorescence, which reports on probe
clustering and hairpin unfolding, while only a ∼2.5-fold
enhancement was observed in the A488 channel, which
exclusively reports on clustering (Figure 1C). Fluorescence
from both channels colocalized with the RICM interference
pattern and were most pronounced at the center of the
microparticle-SLB contact zone, confirming microparticledriven probe clustering (Figure 1D).
To estimate the relative fraction of open hairpins induced by
microparticle binding, we created normalized Cy3B and A488
images (normalized to a region of interest lacking cells). The
normalized Cy3B image was divided by the normalized A488
image, and then was subtracted by 1 to obtain a tension density
map, such that a tension density signal of ∼0 corresponds to
the background (Scheme 1 and Figure S3 for detailed image
processing procedures). Tension density signals that are greater
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Figure 3. (A) Representative images (RICM, Cy3B, A488, and tension density) of CD4+ T-cells plated on fluid SLBs containing tension probes
(upper panel) or control duplexes (lower panel) for a duration of 30 min. (B) Scatter plot showing the mean tension density signal generated on
tension probes and control duplexes within the cSMAC structure (n = 25 cells). (C) Representative images (RICM, Cy3B, A488, and tension
density) of CD4+ T-cells plated on the hindered SLB displaying tension probes. The mobility of tension probes was limited due to the high density
of streptavidin on the SLB. (D) Line profile across the cell (dashed line in panel C) showing differential response in the Cy3B and A488 channels.
(E) Representative images (RICM, Cy3B, A488, tension density, and zoom in) of T-cells pretreated with 50 μM blebbistatin (bleb) or without
blebbistatin (control) and plated onto the 4.7 pN tension probe surface for a duration of 30 min. Scale bars = 5 μm.
26.4%) in the Cy3B channel, whereas tension probes
immobilized on glass surfaces had an average of 4-fold
enhancement in Cy3B fluorescence (400 ± 115%) underneath
the microparticle-surface contact zone (Figure S5). These
results show that ratiometric tension probes can be used to
distinguish signals due to tension from that of clustering.
To test whether the ratiometric tension probe is suitable for
mapping TCR tension and lateral transport (Scheme 1), we
tethered the F1/2 = 4.7 pN probes onto SLBs presenting biotin
and Ni-NTA (0.1% biotin-DPPE, 4% Ni-NTA DOGS and
95.9% DOPC). In a one-pot incubation, we decorated the
tension probes with anti-CD3 antibody that binds and activates
the TCR and also introduced His6-ICAM-1 on the lipid
membrane to support T-cell spreading (Scheme S1 and
Materials and Methods). Given that each gold nanoparticle
presents ∼38 biotinylated DNA hairpins, it is possible for each
particle to bind multiple streptavidin molecules on the SLB,
thus reducing the probe mobility. To minimize multivalent
binding, we tuned the biotin doping level in the SLB (from
0.001% to 1% biotin) and measured the stoichiometry between
the gold particles and streptavidin. We identified that a
concentration of 0.1% biotin-DPPE provides the highest
density and optimal coverage of probes while maintaining
their long-range fluidity (Figure S6). The mobility of the
tension probes and A488-labeled streptavidin on this surface
than 0 indicate a relative fraction of hairpins in the open state.
Molecular binding between microparticles and hairpin tension
probes within the contact zone generated fluorescence
enhancement greatest at the center and had an average tension
density value of 4.29 ± 1.16 (Figure 1E). As a control, we used
AuNP-duplexes lacking a hairpin loop and for which the
fluorescence increase is exclusively due to clustering rather than
hairpin unfolding (see Table S1 for DNA sequences). As
expected, tension density signal on these surfaces were
significantly lower (0.05 ± 0.13) compared to the surfaces
decorated with hairpin tension probes (Figure S4 and Figure
1E). Collectively, these results indicate that the ratiometric
response of the tension probe is largely due to mechanical
unfolding of the hairpin stem-loop of the tension probes.
We further evaluated the interaction between the microparticles and tension probes by immobilizing the tension
probes on glass surfaces where probe clustering is prohibited
(Scheme S2). On these surfaces, the Cy3B signal was localized
at the edges of the microparticle-surface junction. Unlike SLB
surfaces, line scan analysis across the microparticles revealed
two local maxima in Cy3B channel, and the peaks encased the
center of microparticle-surface contact zone (Figure S5). In
contrast, no accumulation of fluorescence in the A488 channel
was observed (Figure S5). Control experiments using DNA
duplexes showed a small increase in fluorescence (25.5 ±
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was confirmed by fluorescence recovery after photobleaching
(FRAP) experiments. Both the Cy3B and A488 fluorescence
channels showed ∼90% recovery within 90s (Figure 1F). The
estimated lateral diffusion coefficients (D) of the fluorescent
streptavidin and tension probes are almost identical (∼1 μm2/
s), confirming that the tension probes are primarily bound to
streptavidin tethered on the lipid membrane (Figure 1G).
With the fluid AuNP-tension probes in hand, we next plated
CD4+ T-cells directly onto the surface. T-cells rapidly spread
upon initial engagement to the SLB (within the first 3 min). In
a representative cell shown in Figure 2A, time-lapse imaging
showed the accumulation of fluorescence in both Cy3B and
A488 channels underneath the cell (Figure 2A, t = 3 min, white
circle). The tension density (ratiometric) signal showed a
gradual increase in intensity that colocalized with a subset of
the accumulating Cy3B signal. This indicates that TCRs
transmit mechanical forces exceeding F1/2 = 4.7 pN to a fraction
of the clustering anti-CD3 ligands. The T-cell continuously
translocated anti-CD3 probes throughout the 15 min duration
of the video. Starting at t = 4 min, centripetal movement of
clusters was accompanied by waves of inward tension density
signal (Figure 2A and Supporting Movie 1). A larger fraction of
tension probes were unfolded at the periphery of the
accumulating clusters (Supporting Movie 1). Kymographs of
different region of interests (ROIs) showed that tension
gradually developed across the cell surface over the 15 min
duration of the video, and TCR tension and clustering are
closely linked in space and time (Figure 2B). Note that the
tension density signal was most pronounced for larger clusters
at the micron-scale and this signal was highly dynamic. Smaller
oligomers or monomers may also experience mechanical strain
that is not reported by our probes because it is below the 4.7
pN threshold for DNA unfolding or possibly due to the low
signal-to-noise ratio associated with smaller assemblies. The
lateral translocation of TCR-ligand complexes and their
accumulation at sites of diffusion barriers strongly suggests
that these complexes experience mechanical strain.24 Nonetheless, our results provide the first direct evidence that the TCR
transmits pN mechanical strain to its ligand within a fluid
intermembrane junction.
We next aimed to investigate whether TCR-ligand complexes
experience tension within the cSMAC, which forms at later
points after formation of the immunological synapse and is
associated with signal termination and receptor degradation.13,55 An important question pertains to the cSMAC
structure is whether TCR-ligand complexes in this centralized
assembly experience mechanical load during TCR recycling.56
To answer this question, T-cells were incubated with the
tension probe surfaces for 30 min to allow for complete
cSMAC formation. After 30 min of cell spreading, we observed
Cy3B and A488 signal in a central region that is a hallmark
feature of cSMAC formation (Figure 3A).57 The cSMAC
displayed strong tension density signal with a maximum value
of ∼2, exceeding the values observed during initial TCR-ligand
binding and clustering (Figure 3A). Also, the tension density
within this structure was more homogeneous and less dynamic.
Control experiments that used DNA duplexes showed the
accumulation of anti-CD3 probes within a central cluster but
did not display significant tension density signal (Figure 3A).
Scatter plot analysis revealed that an average tension density
signal of 0.37 ± 0.31 within the cSMAC while the control
duplexes only had negligible signal (−0.02 ± 0.08) (n = 25 cells
for each group, Figure 3B). Immunostaining further confirmed
that the Cy3B and A488 signals are strongly associated with
TCR (Pearson correlation coefficients of ∼0.8, Figure S7).
Taken together, these experiments demonstrate that the TCRligand complexes experience significant tension within the TCR
recycling cSMAC structure.
To investigate the role of long-range lateral mobility of the
ligand and how this influences TCR force transmission, we
limited the mobility of SLBs by increasing streptavidin density
(using 4% biotin-DPPE lipid composition). Lateral diffusion on
these surfaces was significantly reduced as shown by FRAP
measurements, in which the Cy3B signal from tension probes
showed only ∼40% recovery after 15 min (Figure S8). The
reduced mobility could be due to local molecular crowding of
the lipid bound streptavidin and the tension probes. T-cells
plated on these surfaces showed reduced ligand translocation
and accordingly, cSMAC formation was inhibited. Nonetheless,
T-cells plated on these surfaces showed that TCR-ligand
complexes experienced tension across the cell junction and
preferentially at the cell perimeter (Figure 3C and D). This
spatial distribution of TCR tension is similar to that obtained
using immboile tension probes.32 Line scan analysis across the
cell-SLB contact revealed that Cy3B signal exceeded the A488
signal across the whole intermembrane junction (Figure 3D).
These results demonstrate that T-cells transmit pN forces to
ligand−receptor complexes with highly hindered mobility.
Literature precedent revealed that pretreatment of T-cells
with blebbistatin not only retarded their ability to form the
cSMAC58,59 but also reduced IL-2 cytokine production.60
These observations identify myosin IIA as an essential
component contributing to TCR transport and ultimately Tcell immune function. To investigate whether impairment of
myosin IIA activity directly regulates TCR forces, we pretreated
cells with 50 μM blebbistatin and plated these cells onto SLBs
modified with tension probes for 30 min to allow for cell
spreading. Under these conditions, T-cells formed limited
clusters rather than the cSMAC (Figure 3E, A488 channel) and
the tension density signal was dissipated (Figure 3E). This
result shows that myosin IIA activity is required for mounting
TCR tension during receptor clustering.
In summary, we report the general design of ratiometric
tension probes for direct imaging of mechanical tension
experienced by ligand−receptor complexes within intermembrane junctions. Our ratiometric tension probes showed pN
tension experienced by TCRs undergoing clustering and
translocation in a myosin IIA dependent fashion. We also
revealed mechanical forces within the cSMAC, which is
possibly associated with the endocytosis of TCRs for recycling.
Our approach is broadly applicable for studying the interplay
between force and receptor clustering for juxtacrine receptor
signaling pathways such as those for B-cell receptors, Eph−
ephrin, cadherins, and Notch−Delta.
■
ASSOCIATED CONTENT
S Supporting Information
*
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.nanolett.6b01817.
DNA sequences used in this study, supporting schemes
for fabrication of surfaces, and supplementary data.
(PDF)
Tracking TCR cluster formation and tension. Representative time-lapse video of a CD4+ T-cell plated on a SLB
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presenting tension probes and ICAM-1 adhesion
molecules. RICM (Top left), A488 (clustering, top
right) are shown in the top panel. Cy3B (clustering and
tension, bottom left) and tension density (bottom right)
are shown in the bottom channel. The duration of the
movie is 15 min. Scale bar = 5 μm.(AVI)
AUTHOR INFORMATION
Corresponding Author
*E-mail: [email protected].
Author Contributions
V.P.-Y.M. and Y.L. contributed equally.
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
K.S. would like to thank for the financial support from the NIH
(R01-GM097399), the Alfred P. Sloan Research Fellowship,
the Camille-Dreyfus Teacher-Scholar Award, and the NSF
CAREER Award (1350829). L.B. was supported by a
postdoctoral fellowship from the National Multiple Sclerosis
Society (F1963A1/1). B.E. was supported by NIH Grants R01AI096879.
■
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